Confocal fluorescence microscopy is a technology that is capable of obtaining an improved image by removing background signals emitted from parts other than the focal plane of a microscope lens using a pinhole or the like, compared to conventional fluorescence microscopy. For this reason, confocal fluorescence microscopy technology is widely used in biological research.
A common confocal fluorescence microscopy technology requires scanning in two axial directions in order to obtain one image because light is focused in a dot form in order to observe fluorescent light. Accordingly, the common confocal fluorescence microscopy technology is disadvantageous in that it takes an excessively long time to obtain one image and it is difficult to observe the rapid movements of biological molecules and interactions therebetween within a cell.
A method devised to overcome the above disadvantages is line scan confocal microscopy or spinning disk confocal microscopy. In line scan confocal microscopy, light is focused in a line form, rather than in a dot form, using a cylindrical lens. As a result, the time it takes to obtain an image can be significantly reduced because scanning only in a single axial direction is required to obtain an image. In spinning disk confocal microscopy, an image can be rapidly obtained in such a way as to divide light into several parts, form several dot-shaped focuses at the same time, bore a hole through a disk, and rotate the disk. These two methods can obtain images at a speed (of several tens of Hz) close to a real-time speed.
Recently, with the advancement of related technology, the fluorescence microscopy technology has been advanced to the extent that a single fluorescent molecule can be observed. As a single fluorescent molecule can be observed, the various phenomena of life that were difficult to observe have become understood, and thus the fluorescence microscopy technology is being widely used. A single fluorescent molecule can be observed only when background signals are maximally reduced because the single fluorescent molecule has a very weak fluorescent signal. For this purpose, a total internal reflection method is widely used. When total reflection is generated between a slide and a medium, an evanescent wave is generated, which excites a fluorescent light substance. In this case, background signals generated from parts other than a part where total reflection is generated can be reduced because the range of the evanescent wave is very short, that is, about 200 to 300 nm from a surface where total reflection was generated. However, phenomena occurring inside a cell cannot be observed using the total internal reflection method because only fluorescent light in a very short range near a surface of the cell can be observed using the total internal reflection method.
The confocal microscopy method can be used to measure fluorescent light at a deep location inside a cell and the signal of a single fluorescent molecule, unlike the total reflection microscopy method. However, a common confocal microscope may not be used to observe a rapid change within a cell because the time it takes to obtain an image is long, as described above. In particular, because of its low speed, it is difficult to apply the common confocal microscope to recent ultra-high definition imaging technology based on the determination of the location of a single fluorescent molecule. In the case of line scan confocal microscopy and spinning disk confocal microscopy used to overcome measurement speed issues, the degree to which background noise is reduced is lower than when using a dot-shaped confocal method, and a detector used for observation has low performance. Accordingly, a single fluorescent molecular signal cannot be observed using currently commercialized equipment.
As a result, in order to observe a single molecule fluorescent light in a thick object, such as a cell or a tissue, there is a need for 1) an ability to observe fluorescent light at a deep location, 2) a rapid measurement speed of several tens of Hz or higher, and 3) a high signal-to-noise ratio and high-performance detector for observing single molecule fluorescent light.